Transcriptional profiling reveals molecular signatures associated with HIV permissiveness in Th1Th17 cells and identifies Peroxisome Proliferator-Activated Receptor Gammaas an intrinsic negative regulator of viral replication

Abstract

BackgroundWe previously demonstrated that primary Th1Th17 cells are highly permissive to HIV-1, whereas Th1 cells are relatively resistant. Molecular mechanisms underlying these differences remain unknown.ResultsExposure to replication competent and single-round VSV-G pseudotyped HIV strains provide evidence that superior HIV replication in Th1Th17 vs. Th1 cells was regulated by mechanisms located at entry and post-entry levels. Genome-wide transcriptional profiling identified transcripts upregulated (n = 264) and downregulated (n = 235) in Th1Th17 vs. Th1 cells (p-value < 0.05; fold change cut-off 1.3). Gene Set Enrichment Analysis revealed pathways enriched in Th1Th17 (nuclear receptors, trafficking, p38/MAPK, NF-κB, p53/Ras, IL-23) vs. Th1 cells (proteasome, interferon α/β). Differentially expressed genes were classified into biological categories using Gene Ontology. Th1Th17 cells expressed typical Th17 markers (IL-17A/F, IL-22, CCL20, RORC, IL-26, IL-23R, CCR6) and transcripts functionally linked to regulating cell trafficking (CEACAM1, MCAM), activation (CD28, CD40LG, TNFSF13B, TNFSF25, PTPN13, MAP3K4, LTB, CTSH), transcription (PPARγ, RUNX1, ATF5, ARNTL), apoptosis (FASLG), and HIV infection (CXCR6, FURIN). Differential expression of CXCR6, PPARγ, ARNTL, PTPN13, MAP3K4, CTSH, SERPINB6, PTK2, and ISG20 was validated by RT-PCR, flow cytometry and/or confocal microscopy. The nuclear receptor PPARγ was preferentially expressed by Th1Th17 cells. PPARγ RNA interference significantly increased HIV replication at levels post-entry and prior HIV-DNA integration. Finally, the activation of PPARγ pathway via the agonist Rosiglitazone induced the nuclear translocation of PPARγ and a robust inhibition of viral replication.ConclusionsThus, transcriptional profiling in Th1Th17 vs. Th1 cells demonstrated that HIV permissiveness is associated with a superior state of cellular activation and limited antiviral properties and identified PPARγ as an intrinsic negative regulator of viral replication. Therefore, triggering PPARγ pathway via non-toxic agonists may contribute to limiting covert HIV replication and disease progression during antiretroviral treatment.

HIV-1 selectively targets gut-homing CCR6+CD4+ T cells via mTOR-dependent mechanisms

“…Although the T cell activation marker CD38 was similarly upregulated by ATRA in both subsets (Supplemental Figure 3), transcripts for MAP3K4, a Th17-specific kinase (54) we previously identified as a HDF (20), were expressed at superior levels and upregulated by ATRA in CCR6 + versus CCR6 -T cells (Supplemental Figure 3). Moreover, the transcripts for PPARγ, a transcriptional repressor of RORγt (55) and Th17 intrinsic negative regulator of HIV replication (19), were downregulated by ATRA specifically in CCR6 + T cells (Supplemental Figure 3). Furthermore, CCR6 + versus CCR6 -T cells stimulated via the TCR in the presence/absence of ATRA expressed decreased levels of IFN-stimulated genes, such as IFITM1, IFITM2, IFITM3, and IRF8 (Supplemental Tables 1-4), consistent with the low Th17 ability to respond to IFN (23).…”

“…The full-length T/F THRO HIV-1 Infectious Molecular Clone (catalog 11919) was obtained from John Kappes through the NIH AIDS Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, NIH (44). HIV stocks were produced, titrated, and used to infect cells (25-50 ng HIV-p24/10 6 cells), as previously described (16,19,20,36,42). Quantification of integrated and Gag HIV DNA.…”

“…Quantification of integrated and Gag HIV DNA. Levels of integrated and Gag HIV DNA were quantified in cell lysates by ultrasensitive nested real-time PCR (10 5 cells/test in triplicates; detection limit, 3 HIV DNA copies), as previously described (16,19,20,36,37,42,93).…”

“…+ T cells are highly permissive to infection (16)(17)(18) as a consequence of their expression of HIV dependency factors (HDFs) acting at entry and postentry levels (19,20), combined with the lack of HIV restriction mechanisms Gut-associated lymphoid tissues are enriched in CCR6 + Th17-polarized CD4 + T cells that contribute to HIV-1 persistence during antiretroviral therapy (ART). This raises the need for Th17-targeted immunotherapies.…”

Antibody persistence and T-cell balance: Two key factors confronting HIV vaccine development

“…[39][40][41]. Of these lineages, only Th17 cells are readily infected with R5 HIV (42,43), and they are the major CD4 + T-cell lineage at mucosal sites (reviewed in ref. 44).…”